The relationship between nucleotide sequence and gene function will be explored for the set of yeast tRNA Tyr genes. A combination of formal genetic techniques and DNA biochemistry will be employed. These eight unlinked genes, which can all mutate to nonsense suppressors, are readily manipulated genetically. They have already been cloned and several of them have been sequenced so the groundwork for an integrated molecular genetic analysis is already laid. Two basic types of questions will be examined. First, the role of non-coding sequences in gene function will be explored. Specifically, the role of sequences within the promoter, the transcribed leader, the intervening sequence, and the terminator will be studied both by characterizing loss-of-function mutations within these regions and by testing the functionality of genes that have been mutagenized in vitro. These in vivo tests of the functionality of altered genes will be carried out using a yeast transformation system. Secondly, the molecular basis of the evolutionary stability of this gene family will be studied by attempting to determine the frequency at which sequence information is exchanged between the different loci. The hypothesis that homology through the intervening sequences is a prerequisite to inter-locus genetic exchanges will be directly tested using genes that have had their intervening sequences removed in vitro.